The aim of this proposal is to study the mechanism of induction of the c-fos and c-jun proto-oncogenes by growth factors. They are regulated by distinct pathways and sequence elements. The study of regulation of these genes should lead to an understanding of how these genes are switched on and off and to the identification of additional components of signaling pathways critical for stimulation of cell growth. The c-fos promoter is regulated by growth factors through the SRE site. Pathways to this site lead to phosphorylation of the SRF- associated TCF proteins but serum and other factors also activate the SRE independently of TCF by an unknown mechanism. The most likely regulatory mechanism involves additional complexing proteins. One recently identified SRF-binding protein is the bZIP factor ATF6. Inhibition of ATF6 levels decreased serum induction of the SRE. In addition, GALA-ATF6 can be regulated by p38 MAPK pathways such that it could participate in regulation. DNA binding, heterodimerization and regulation of ATF6 will be characterized. ATF6 and/or a putative partner will be tested for roles in growth factor regulation of the c-fos gene. Since the known SRF-binding proteins do not appear to be required for serum regulation of the SRE we will identify novel SRF-complexing proteins. A factor identified in gel mobility shift assays will be purified and cloned and a novel mammalian two-hybrid screen will be used to identify additional SRF-complexing proteins involved in serum regulation of c-fos. EGF induction of the c-jun promoter is controlled by MEF2 and ATF sites. These sites are regulated by a ras to rac to MEKK pathway. How this pathway directly regulates the c-jun promoter through the MEF2 and ATF sites will be studied. In HeLa cells MEF2D binds to the MEF2 site and GALA-MEF2D is regulated by MEKK. Preliminary results indicate that EGF induces MEF2D phosphorylation. The regions of MEF2D required for its regulation and phosphorylation will be mapped. If phosphorylation is required, we will identify the MEF2D-protein kinase. ATF1 binds to the c-jun ATF site in HeLa cells and EGF induces ATF1 phosphorylation. The importance of this phosphorylation for c-jun regulation will be tested and the protein kinase responsible for EGF-induced ATF1 phosphorylation will be identified.